Cardiac muscle protein analysis by high-resolution and microscale two-dimensional gel electrophoresis

An improved method of two-dimensional gel electrophoresis of small muscle samples is described. Isoelectric focusing of cardiac whole muscle homogenate in agarose gels containing urea and detergent has a markedly increased resolution. Equilibration of the first-dimensional gels with detergent before application to the second-dimensional gels is unnecessary in this system. By applying this method to rat cardiac whole muscle, high-molecular weight proteins, such as myosin heavy chains, are focused on the first-dimensional gels and, in addition, minor components are resolved on the second-dimensional gels, without loss during equilibration with detergent. The two-dimensional electrophoretic patterns of rat cardiac whole muscle obtained with this method reveal numerous clearly separated spots. By analyzing the two-dimensional electrophoretic patterns of rat cardiac whole muscle and various rat cardiac fractions, and by staining the calcium-binding proteins with "Stains-all", we identified some cardiac muscle components, such as myosin heavy and light chains, actin, tropomyosin, and troponin C, but additional work is required to identify the remaining spots. The two-dimensional electrophoretic system described here makes possible the effective resolution of whole cardiac muscle homogenate from small samples, and looks promising as an aid to muscle research.     

A high resolution two-dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins.

An improved two-dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two-dimensional electrophoretic patterns afford much information of clinical significance. However, current two-dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two-dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two-dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two-dimensional gels and is no more laborious than current techniques.     

GELANAL, a personal computer-program to compare protein patterns on two-dimensional polyacrylamide gels

Comparing and analyzing a series of two-dimensional gels by hand is troublesome and subjective. So far a number of systems for automatic analysis have been developed on mainly mainframe computers, using complex algorithms. This paper presents an inexpensive system, based on a simple Pascal program, to compare individual spots on two-dimensional gels using an IBM or compatible personal computer in a qualitative way. The accuracy of the method is demonstrated by comparing two patterns of the same extract from different runs.     

Rapid protein identification using a disposable on-line clean-up/concentrating device and electrospray ionization mass spectrometry

A simple, low-cost, expedient method has been developed for identification of proteins isolated from two-dimensional (2D) gels. The method described uses a disposable on-line clean-up device, a syringe infusion pump and electrospray ionization mass spectrometry (ESI-MS). The on-line clean-up and concentrating device is a tapered capillary column filled with 1.5 cm of 5 microm C18 particles. The short column was easily prepared and was connected directly to the ESI source through a low-flow ESI sprayer. Peptides resulting from enzymatic digestion of proteins were eluted from the short column isocratically using a syringe infusion pump and analyzed by ESI-MS. This simple set-up was found useful in the analysis of proteins isolated from 2D gels. Compared to the more conventional micro-liquid chromatography/tandem mass spectrometry (microLC/MS/MS), this method can identify proteins rapidly without the need for an HPLC pump and removes the problem of cross-contamination caused by system carryover. These advantages make the method described competitive with conventional LC/MS even though the latter method gives slightly expanded sequence coverage.     

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.     

Rapid imaging, using a cooled charge-coupled-device, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first-dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone

A new method for visualising proteins in two-dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) while present in the first-dimensional gel after isoelectric focusing and subsequently electrophoresed into the second-dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge-coupled-device was used to view the two-dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two-dimensional gels at the end of electrophoresis with no further processing.     

Explorative data analysis of two-dimensional electrophoresis gels

Methods for classification of two-dimensional (2-DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two-dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening of gels is presented. First, an approach is demonstrated in which no prior knowledge of the separated proteins is used. Alignment of the gels followed by a simple transformation of data makes it possible to analyze the gels in an automated explorative manner by principal component analysis, to determine if the gels should be further analyzed. A more detailed approach is done by analyzing spot volume lists by principal components analysis and partial least square regression. The use of spot volume data offers a mean to investigate the spot pattern and link the classified protein patterns to distinct spots on the gels for further investigation. The explorative approach in analysis of 2-D gels makes it possible, in a fast and convenient way, to screen many gels in order to determine the protein patterns that form clusters and could be selected for further examination.     

Identification of nolR-regulated proteins in Sinorhizobium meliloti using proteome analysis

Extractable proteins from Sinorhizobium meliloti strains AK631 and EK698 (a Tn5-induced noIR-deficient mutant of AK631), grown in tryptone agar (TA) medium with or without the addition of the plant signal luteolin, were separated by two-dimensional gel electrophoresis and compared. Analysis of silver-stained gels showed that the noIR mutant had 189 proteins that were significantly altered in their levels (101 protein spots up- and 88 downregulated). Coomassie-stained preparative two-dimensional (2-D) gels or polyvinylidene difluoride (PVDF) membranes blotted from preparative gels showed that at least 52 of the altered proteins could be reproducibly detected and isolated from the noIR mutant. These 52 altered protein spots were classified into five groups based on an assessment of protein abundance by computer analysis and the effect of the presence or absence of luteolin addition to the growth medium. N-terminal microsequencing of 38 proteins revealed that the most striking feature of the consequence of the noIR mutation is the number and broad spectrum of cellular functions that are affected by the loss of the NoIR function. These include proteins involved in the tricarboxylic acid (TCA) cycle, heat shock and cold shock proteins, protein synthesis, a translation elongation factor, oxidative stress and cell growth and maintenance functions. We propose that the NoIR repressor is a global regulatory protein which responds to environmental factors to fine-tune intracellular metabolism.     

Image analysis techniques for automatic evaluation of two-dimensional electrophoresis.

Techniques for automatic analysis of two-dimensional electrophoresis gels by computer-aided image analysis are described. Original gels or photographic films are scanned using a laser scanner and the files are transferred to a microcomputer. The program package first performs a compression and preevaluation of the files. Spot identification and quantification is performed by the chain code algorithm after appropriate zooming and cutting. Labeling facilitates spot identification and quantification in numerical and graphical (pseudocolor) representation on peripheral devices for camera ready output. Interpolation between measured basepoints is performed by cubic spline algorithms which are automatically switched on and off, depending on the need by the program. High speed analysis and graphic representation is achieved using fast Assembler language routines rather than high level languages. One-dimensional gels can be analyzed using the same software. Spot matching between parallel two-dimensional gels has not yet been implemented.     

Two-dimensional gel electrophoresis, protein electroblotting and microsequencing: a direct link between proteins and genes.

We have used two-dimensional gel electrophoresis as a general "preparative" method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4-vinyl-N-methylpyridinium iodide)-coated glass fiber sheets. The major spots were excised and subjected to NH2-terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two-dimensional gels and the Coomassie Brilliant Blue-stained spots were excised from the gels. The combined spots were re-eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase-high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two-dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.     

Peptide mapping of polypeptides separated by two-dimensional electrophoresis: protease digestion directly on the two-dimensional gel followed by electrophoresis in reverse direction

A method is described which allows to reveal simultaneously the proteolytic patterns of numerous polypeptides separated by two-dimensional electrophoresis. After two-dimensional electrophoresis, the gels were dipped successively in buffers for preequilibration, protease digestion, and reequilibration. They were then returned to the electrophoresis tank, and electrophoresis was continued for a short time. After silver staining, digestion products appeared, lined up behind the original polypeptide spots. The method allows proteolytic patterns of numerous polypeptides to be visualized simply and quickly. Among proteins of wheat leaves, 31 groups of related polypeptides were found according to the similarity of their proteolytic patterns.     

Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins.

The number of unique protein species in proteomes from a single mammalian cell type is not well defined but is likely to be at least 10000-20000. Since standard-size two-dimensional gels typically resolve only about 1500 to 3000 spots, they merely analyze a small portion of these proteomes. In addition, all insoluble proteins and typically proteins > 100 kDa are seldom resolved on two-dimensional (2-D) gels. The current study demonstrates the feasibility of an overall strategy for more comprehensive quantitative comparisons of complex proteomes derived from physiological fluids or mammalian cell extracts. A key feature of this approach is to prefractionate samples into a few well-resolved fractions based on the proteins' isoelectric points (pIs) using microscale solution isoelectric focusing. These fractions are then separated on narrow pH range two-dimensional gels approximately +/- 0.1 pH unit wider than the prefractionated pool. When this prefractionation approach is applied to complex mammalian proteomes, it improves resolution and spot recovery at high protein loads compared with use of parallel narrow pH range gels without prefractionation. The minimal cross-contamination between fractions allows quantitative comparisons in contrast to most alternative prefractionation methods. In addition, complementary data can be obtained by parallel analysis of the solubilized fraction on high-resolution large-pore-gradient one-dimensional gels followed by mass spectrometric identification to analyze proteins between 100 and approximately 500 kDa. Similarly, insoluble proteins can be analyzed using large-pore gels for large proteins and 10-12% one-dimensional sodium dodecyl sulfate (SDS) gels for smaller proteins. Together, these strategies should permit more reliable quantitative comparisons of complex mammalian proteomes where detection of at least 10000 protein spots is needed in order to analyze the majority of the unique protein species.     

Determination of protein spots separated by two-dimensional polyacrylamide gel electrophoresis

A simple method for quantitating proteins in the spots on two-dimensional polyacrylamide gel electropherograms is described. The system consists in three steps: (1) O'Farrell's two-dimensional gel electrophoresis of the proteins to be analysed; (2) staining of the gels with Coomassie brilliant blue; and (3) determination of the area and integrated density of the stained spots by the Joyce Loebl Magiscan-1 image analysis system. The method can be used for the determination of proteins in the range 0.5-100 micrograms/cm2; the amount of protein involved in most spots detected by the staining method actually falls within this range. As the minimum spot diameter that can easily be handled by the method is about 2 mm, as much as 30 ng of protein in such a spot can be determined. The method can also be applied to autoradiograms.     

Chromatographic and electrophoretic studies of circulating immune complexes in plasma

The protein nature of soluble immune complexes from fresh plasma was studied by combining several analytical biochemical techniques. Free immunoglobulins (Ig) G were separated from larger immune complexes by gel permeation chromatography. In a second step, immune complexes, free IgA and IgM were isolated by protein-A and protein-G affinity chromatography and analysed by two-dimensional gel electrophoresis. Sixteen plasma samples from healthy donors were analysed and evaluated visually. Their protein profiles on the gels turned out to be similar, showing only slight quantitative differences. In one case, additional proteins were detected. To prove the ability of the method, immune complexes were analysed from four plasma samples that showed macro creatine kinase type 1, a complex formation between creatine kinase BB and IgG. This methodology can be used for the examination of immune complexes of unknown protein composition in serum or plasma.     

Production of high purity proteins by elution of individual fractions from two-dimensional gels.

Results are presented on the production of purified myocardial proteins from two-dimensional (2-D) gels. Proteins were fixed in a native condition using potassium acetate and then eluted into an aqueous solution. Homogeneous tropomyosin and myosin light chain fractions and a number of nonidentified myocardial proteins present on 2-D gels were obtained.     

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.     

Electrophoretic heterogeneity of maize histones

Histones from maize embryos and seedlings have been isolated using a fast extraction procedure. Three different electrophoretic systems have been applied for the study of the heterogeneity of maize core histones. Electrophoresis in acetic acid/urea polyacrylamide gels, containing high concentrations of urea, resulted in optimum fractionation of the core histones and especially of histone H4. Sodium dodecyl sulfate-containing polyacrylamide gels were not useful for the fractionation of maize histone classes H2a and H2b, nor for the various subfractions of H3 and H4. Gels containing Triton X-100, used for the dimension in two-dimensional electrophoresis proved to be efficient for the separation of all histone classes, as well as their structural variants and chemical modifications. Maize core histones have been oxidized in an attempt to define which of the Triton X-100 resolved subfractions represent oxidation forms.     

Diagnostic use of an analysis of urinary proteins by a practicable sodium dodecyl sulfate-electrophoresis method and rapid two-dimensional electrophoresis

Two methods suitable for routine clinical analyses of urinary proteins are presented and compared. The first is a horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique, suitable for simultaneous analysis of 20 native urinary samples. This method uses polyacrylamide gradient gels, prepared with a laboratory-built gel casting device. The second method is a rapid two-dimensional electrophoresis procedure, combining cellulose acetate electrophoresis and sodium dodecyl sulfate-electrophoresis. The first step uses a routine system (Chemetron), the second separation step followed by staining with Coomassie Brilliant Blue R is performed on the PhastSystem. The resulting two-dimensional patterns reveal urinary proteins distributed according to the 5-zone pattern of native proteins (albumin, alpha-1, alpha-2, beta, gamma-globulin) as well as to the logarithm of their molecular weights. Examples of (routine) diagnoses with a special interest in the monitoring of kidney transplant patients are shown.     

Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE

Horizontal nondenaturing electrophoresis of proteins in polyacrylamide gels was used to observe specific interactions between membrane proteins. The method was particularly well suited for solubilized transporters of the outer membrane of Gram-negative bacteria, and allowed specific complexes of transporter and the inner-membrane protein TonB to be isolated. We have used this method to investigate the interactions between four different outer-membrane transporters, and the TonB proteins from two different organisms. The results show that a stable complex can be isolated on gels for all the proteins studied, but can depend in some cases of the detergent used for solubilization. Furthermore, we observe cross-species interaction as TonB from a given organism can interact with transporters from another organism.     

Enhanced Protein Extraction from Tobacco Roots for Proteomic Analysis

Plant roots contain low protein concentrations and many interferences for protein extraction and two-dimensional electrophoresis analysis. Therefore, the extraction of high-quality protein from tobacco roots for proteomic analysis is a challenge. Three protein extraction methods (the trichloroacetic acid-acetone, phenol extraction, and trichloroacetic acid-acetone-phenol methods) for tobacco root proteins were compared using protein yields, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional electrophoresis. The trichloroacetic acid-acetone-phenol method provided a higher spot resolution (505 ± 18 spots), the least streaking, and larger protein yields (2200 ± 20 µg/g fresh weight) on two-dimensional electrophoresis gels for tobacco roots, and hence is the most suitable method for the characterization of tobacco roots.